The broad overall objective of this competing renewal grant application is to develop novel prodrug strategies to improve ocular antiviral drug therapy, in the treatment of human cytomegalovirus (HCMV) retinitis. In the previous grant period, microdialysis technique has been utilized to study ocular pharmacokinetics in anesthetized and conscious animal models. Retinal drug delivery may be enhanced by exploiting the membrane transporters on the neural retina, RPE and/or endothelial cells of the retinal blood vessels. We propose to synthesize a series of dipeptide, amino acid and folate mono- and di- ester prodrugs of ganciclovir (GCV) to target peptide, amino acid and folate transporters/receptors respectively. The proposed prodrugs would not only improve the ocular bioavailability of GCV, but also may exhibit diminished cytotoxicity, require lower doses, and a decreased frequency of administration. By simultaneous targeting of multiple transporters having no overlapping substrate specificity, we can achieve higher intracellular concentrations of GCV due to enhanced uptake of the prodrugs followed by enzymatic conversion in the retinal cells. The specific aims of this renewal application are: 1. To synthesize mono- and di-ester derivatives of GCV targeting peptide transporters - Val-Val-GCV, Val-Gly-GCV, Gly-Val- GCV, Gly-Tyr-GCV and Val-Tyr-GCV; amino acid transporters - gamma,-Glu-GCV, Phe-GCV, Tyr-GCV and Trp-GCV; and folate transport systems - folate ester of GCV. 2. To determine antiviral efficacy and cytotoxicity of the proposed compounds against in vitro viral screens of HCMV, HSV-1, HSV-2, VZV, and EBV and to conduct in vivo efficacy studies against HCMV retinitis in SCID mouse model, by NIAID supported research at the University of Alabama, Birmingham (P.I. Dr. Earl Kern). 3. To conduct uptake studies of dipeptide, amino acid (targeted to glutamate, LNAA transporters) and folate (targeted to folic acid receptors/transporters) mono- and di-ester prodrugs of GCV, a) in vitro, using ARPE-19 cell line, and b) ex vivo/in vivo, using rabbit retina. Our aim is also to study the retinal concentrations of GCV following simultaneous administration of a prodrug combination targeted towards peptide, amino acid and folate transporters. 4. To evaluate in vivo ocular bioavailability of GCV in the vitreous and anterior chambers utilizing dual probe ocular microdialysis technique following IV and intravitreal administrations. The ocular bioavailability of GCV upon administration of a prodrug combination targeted towards peptide, amino acid and folate transporters will be determined. 5. i) to develop a novel injectable, biodegradable, thermosensitive in situ gel forming system, containing drug and drug loaded microspheres and (ii) to evaluate in vivo ocular bioavailability of GCV with microdialysis technique following episcleral deposition of the gel formulation in a conscious animal model.